Precisely regulated cell proliferation is essential for embryonic development as well as homeostasis in adult organs and tissues, whereas uncontrolled cell proliferation is a hallmark of cancer. Thus, elucidating how the cell cycle machinery is controlled is an important area of research in cancer cell biology. A large body of evidence has established a basic paradigm of the control of cell cycle progression involving the Retinoblastoma (Rb) protein family in conjunction with the E2F family of transcription factors. During G0/G1, interaction of hypo-phosphorylated Rb proteins with E2Fs prevents the transcription of E2F target genes. Cyclin-CDK complexes generated during cell cycle progression hyper-phosphorylate Rb, leading to release of Rb from E2Fs; this allows E2F target gene transcription and cell cycle progression. We have identified the mammalian ortholog of Drosophila ecdysoneless (Ecd) protein as a novel and essential regulator of Rb-E2F-dependent cell cycle progression. Loss of Ecd retards the separation of Rb from E2F, arrests cells at G1/S boundary and prevents cell cycle progression. These findings have led to a new model that represents a fundamental shift in the Rb-E2F-dependent cell cycle control paradigm. Notably, Ecd is overexpressed in breast cancer cell lines as well as in ductal carcinoma in situ and infiltrating ductal carcinomas of the breast. Notably, Ecd overexpression produced two opposite phenotypes: p53-dependent senescence in fibroblasts, compared to rapid transit through cell cycle in immortal human mammary epithelial cells (hMECs) that lack p16; and co-overexpression of Ecd with activated Ras induced a dramatic hyper-proliferation and aberrant branching of hMECs in three-dimensional culture. These features are reminiscent of senescence induced by oncogenes, such as Ras. These findings lead us to hypothesize that Ecd is a novel and essential component of Rb-E2F-dependent control of cell cycle progression, and alterations in the levels and/or function of Ecd contribute to oncogenic transformation. Here, we will address these hypotheses using unique and innovative cellular and animal models established by our team. We will examine the structural basis of the role of Ecd in cell cycle progression and its regulation. We will characterize Ecd-induced cellular senescence. We will analyze the consequences of Ecd overexpression in promoting mammary oncogenesis in vitro and in vivo using inducible transgenic mice. Finally, we will determine if Ecd is essential for mammary tumor initiation, progression and maintenance driven by a human breast cancer-relevant oncogene ErbB2 using mammary-specific deletion of Ecd in Ecd-floxed mice. A successful outcome of our studies will elucidate the role of a novel cell cycle control regulator in breast cancer with broad implications for oncogenesis in human cancer, and could help establish Ecd as a potential therapeutic target in cancer. PUBLIC HEALTH RELEVANCE: This proposal will investigate a new and paradigm-shifting mechanism of how cell cycle is regulated. Ecd, the novel cell cycle regulatory protein under study is overexpressed in human breast cancers and initial studies indicate that forced expression of Ecd in normal breast cells pushes them along tumorigenesis. Our studies are therefore focused on investigating the role of Ecd in breast cancer using unique cellular and animal models specifically created for this project. A successful outcome of our studies will likely have broad implications for understanding mechanisms of human cancer and could help establish Ecd as a potential therapeutic target. While our studies are focused on breast cancer, these studies are likely to be of broad significance for human cancer biology and other diseases given the frequent alterations in cell cycle regulatory mechanisms in human cancers and other pathological conditions.